WO2015187871A1 - Procédés et dispositifs permettant de moduler l'expression génique et l'activité enzymatique - Google Patents

Procédés et dispositifs permettant de moduler l'expression génique et l'activité enzymatique Download PDF

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Publication number
WO2015187871A1
WO2015187871A1 PCT/US2015/034058 US2015034058W WO2015187871A1 WO 2015187871 A1 WO2015187871 A1 WO 2015187871A1 US 2015034058 W US2015034058 W US 2015034058W WO 2015187871 A1 WO2015187871 A1 WO 2015187871A1
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less
micro
amperes
array
reservoirs
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PCT/US2015/034058
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English (en)
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Jeffry Skiba
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Vomaris Innovations, Inc.
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Priority to US15/315,951 priority Critical patent/US20170128720A1/en
Publication of WO2015187871A1 publication Critical patent/WO2015187871A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
    • A61N1/326—Applying electric currents by contact electrodes alternating or intermittent currents for promoting growth of cells, e.g. bone cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/02—Details
    • A61N1/04—Electrodes
    • A61N1/0404—Electrodes for external use
    • A61N1/0408—Use-related aspects
    • A61N1/0468—Specially adapted for promoting wound healing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/02—Details
    • A61N1/04—Electrodes
    • A61N1/0404—Electrodes for external use
    • A61N1/0472—Structure-related aspects
    • A61N1/0476—Array electrodes (including any electrode arrangement with more than one electrode for at least one of the polarities)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/02—Details
    • A61N1/04—Electrodes
    • A61N1/0404—Electrodes for external use
    • A61N1/0472—Structure-related aspects
    • A61N1/0492—Patch electrodes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/02—Details
    • A61N1/04—Electrodes
    • A61N1/05—Electrodes for implantation or insertion into the body, e.g. heart electrode
    • A61N1/0526—Head electrodes
    • A61N1/0548—Oral electrodes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/20—Applying electric currents by contact electrodes continuous direct currents
    • A61N1/205—Applying electric currents by contact electrodes continuous direct currents for promoting a biological process
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
    • A61N1/328—Applying electric currents by contact electrodes alternating or intermittent currents for improving the appearance of the skin, e.g. facial toning or wrinkle treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
    • A61N1/36—Applying electric currents by contact electrodes alternating or intermittent currents for stimulation
    • A61N1/36014—External stimulators, e.g. with patch electrodes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
    • A61N1/36—Applying electric currents by contact electrodes alternating or intermittent currents for stimulation
    • A61N1/36014—External stimulators, e.g. with patch electrodes
    • A61N1/3603—Control systems
    • A61N1/36034—Control systems specified by the stimulation parameters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/02—Details
    • A61N1/04—Electrodes
    • A61N1/0404—Electrodes for external use
    • A61N1/0408—Use-related aspects
    • A61N1/0428—Specially adapted for iontophoresis, e.g. AC, DC or including drug reservoirs
    • A61N1/0432—Anode and cathode
    • A61N1/0436—Material of the electrode
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/02—Details
    • A61N1/04—Electrodes
    • A61N1/0404—Electrodes for external use
    • A61N1/0408—Use-related aspects
    • A61N1/0464—Specially adapted for promoting tissue growth
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/02—Details
    • A61N1/04—Electrodes
    • A61N1/0404—Electrodes for external use
    • A61N1/0472—Structure-related aspects
    • A61N1/0484—Garment electrodes worn by the patient

Definitions

  • the present specification relates to methods and devices useful for modulating gene expression and enzyme activity, as well as disrupting biofilm formation, proliferation, and viability.
  • Such matrices can include a first array comprising a pattern of microcells, for example formed from a first conductive material, the material including a metal species; and a second array comprising a pattern of microcells, for example formed from a second conductive solution, the solution including a metal species capable of defining at least one voltaic cell for spontaneously generating at least one electrical current with the metal species of the first array when said first and second arrays are introduced to an electrolytic solution and said first and second arrays are not in physical contact with each other.
  • an external power source such as AC or DC or mixed AC/DC power, or pulsed RF, or pulsed current, such as high voltage pulsed current.
  • the electrical energy is derived from dissimilar metals creating a battery at each cell/cell interface, whereas those embodiments with an external power source can employ conductive electrodes in a spaced-apart configuration to predetermine the electric field shape and strength.
  • FIG. 1 is a detailed plan view of an embodiment disclosed herein.
  • FIG. 2 is a detailed plan view of a pattern of applied electrical conductors in accordance with an embodiment disclosed herein.
  • FIG. 3 is an adhesive bandage using the applied pattern of FIG. 2.
  • FIG. 4 is a cross-section of FIG. 3 through line 3-3.
  • FIG. 5 is a detailed plan view of an embodiment disclosed herein which includes fine lines of conductive metal solution connecting electrodes.
  • FIG. 6 is a detailed plan view of an embodiment having a line pattern and dot pattern.
  • FIG. 7 is a detailed plan view of an embodiment having two line patterns.
  • FIG. 8 depicts alternate embodiments showing the location of discontinuous regions as well as anchor regions of the wound management system.
  • FIG. 9 (A) is an Energy Dispersive X-ray Spectroscopy (EDS) analysis of Ag/Zn BED ("bioelectric device”; refers to an embodiment as disclosed herein).
  • EDS Energy Dispersive X-ray Spectroscopy
  • FIG. 10 depicts Scanning Electron Microscope (SEM) images of in-vitro Pseudomonas aeruginosa PA01 biofilm treated with placebo, an embodiment disclosed herein ("BED"), and placebo + Ag dressing.
  • SEM Scanning Electron Microscope
  • FIG. 1 1 shows extracellular polysaccharide staining (EPS).
  • FIG. 12 shows live/dead staining.
  • the green fluorescence indicates live PA01 bacteria while the red fluorescence indicates dead bacteria.
  • FIG. 13 shows PA01 staining.
  • FIG. 14 depicts real-time PCR to assess quorum sensing gene expression.
  • FIG. 15 shows electron paramagnetic (EPR) spectra using DEPMPO (a phosphorylated derivative of the widely used DMPO spin trap). Spin adduct generation upon exposure to disclosed embodiments for 40 minutes in PBS.
  • EPR electron paramagnetic
  • FIG. 16 depicts real-time PCR performed to assess mex gene expression upon treatment with Ag/Zn BED and 10mM DTT.
  • FIG. 17 shows Glycerol-3-Phosphate Dehydrogenase (GPDH) enzyme activity.
  • Embodiments disclosed herein include methods, devices and systems that can provide a low level electric field (LLEF) to an area where treatment is desired, such as tissue or an organism (thus a "LLEF system") or, when brought into contact with an electrically conducting material, can provide a low level electric current (LLEC) to an area where treatment is desired, such as tissue or an organism (thus a "LLEC system”).
  • LLEF low level electric field
  • a LLEC system is a LLEF system that is in contact with an electrically- conducting material such as saline or wound exudate.
  • the microcurrent or electric field can be modulated, for example, to alter the duration, size, shape, field depth, current, polarity, or voltage of the system.
  • the watt-density of the system can be modulated.
  • the frequency, phase, amplitude, and wave form can be modulated.
  • Activation gel as used herein means a composition useful for maintaining a moist environment about the area to be treated or improving conductance in the area to be treated.
  • Affixing as used herein can mean contacting a patient or tissue with a device or system disclosed herein.
  • Applied or “apply” as used herein refers to contacting a surface with a conductive material, for example printing, painting, or spraying a conductive ink on a surface.
  • applying can mean contacting a patient or tissue or organism with a device or system disclosed herein.
  • BED or “bioelectric device” as used herein is a LLEC or LLEF system as disclosed herein.
  • Biofilm is any group of microorganisms in which cells adhere to each other, for example on a surface. These adherent cells are frequently embedded within a self-produced matrix of extracellular polymeric substance (EPS).
  • EPS extracellular polymeric substance
  • Biofilm extracellular polymeric substance which is also referred to as "slime” (although not everything described as slime is a biofilm), is a polymeric conglomeration generally composed of extracellular DNA, proteins, and polysaccharides. Biofilms may form on living or non-living surfaces and can be prevalent in natural, industrial and hospital settings.
  • the microbial cells growing in a biofilm are physiologically distinct from planktonic cells of the same organism, which, by contrast, are single-cells that may float or swim in a liquid medium.
  • biofilms including gram-positive (e.g. Bacillus spp, Listeria monocytogenes, Staphylococcus spp, and lactic acid bacteria, including Lactobacillus plantarum and Lactococcus lactis) and gram- negative species (e.g. Escherichia coli, or Pseudomonas aeruginosa).
  • gram-positive e.g. Bacillus spp, Listeria monocytogenes, Staphylococcus spp, and lactic acid bacteria, including Lactobacillus plantarum and Lactococcus lactis
  • gram- negative species e.g. Escherichia coli, or Pseudomonas aeruginosa.
  • Biofilms are highly resistant to antibiotics. Consequently, very high and/or long-term doses are often required to eradicate biofilm-related infections. Biofilms are responsible for diseases, such as:
  • Kidney stones- Biofilms also cause the formation of kidney stones.
  • the stones cause symptoms of disease by obstructing urine flow and by producing inflammation and recurrent infection that can lead to kidney failure.
  • Approximately 15%-20% of kidney stones occur in the context of a urinary tract infection.
  • These stones can be produced by the interplay between infecting bacteria and mineral substrates derived from the urine. This interaction results in a complex biofilm composed of bacteria, bacterial exoproducts, and mineralized stone material.
  • Leptospirosis- Biofilms also cause leptospirosis, a serious but neglected emerging disease that infects humans through contaminated water. Previously, scientists believed the bacteria associated with leptospirosis were planktonic (free-floating).
  • Leptospira interrogans can make biofilms, which could be one of the main factors controlling survival and disease transmission. According to the study's author, 90% of the species of Leptospira tested could form biofilms, and it takes L. interrogans an average of 20 days to make a biofilm.
  • Osteomyelitis- Biofilms may also cause osteomyelitis, a disease in which the bones and bone marrow become infected. This is supported by the fact that microscopy studies have shown biofilm formation on infected bone surfaces from humans and experimental animal models.
  • Biofilms can be formed by bacteria that colonize plants, e.g. Pseudomonas putida, Pseudomonas fluoresceins, and related pseudomonads which are common plant-associated bacteria found on leaves, roots, and in the soil, and the majority of their natural isolates form biofilms.
  • Pseudomonas putida Pseudomonas fluoresceins
  • pseudomonads which are common plant-associated bacteria found on leaves, roots, and in the soil, and the majority of their natural isolates form biofilms.
  • Several nitrogen-fixing symbionts of legumes such as Rhizobium leguminosarum and Sinorhizobium meliloti form biofilms on legume roots and other inert surfaces can be used.
  • Conductive material refers to an object or type of material which permits the flow of electric charges in one or more directions.
  • Conductive materials can include solids such as metals or carbon, or liquids such as conductive metal solutions and conductive gels. Conductive materials can be applied to form at least one matrix. Conductive liquids can dry, cure, or harden after application to form a solid material.
  • discontinuous region refers to a "void" in a material such as a hole, slot, or the like.
  • the term can mean any void in the material though typically the void is of a regular shape.
  • the void in the material can be entirely within the perimeter of a material or it can extend to the perimeter of a material.
  • Dots refers to discrete deposits of similar or dissimilar reservoirs that can function as at least one battery cell.
  • the term can refer to a deposit of any suitable size or shape, such as squares, circles, triangles, lines, etc.
  • the term can be used synonymously with, microcells, etc.
  • Electrode refers to similar or dissimilar conductive materials. In embodiments utilizing an external power source the electrodes can comprise similar conductive materials. In embodiments that do not use an external power source, the electrodes can comprise dissimilar conductive materials that can define an anode and a cathode.
  • Galvanic cell refers to an electrochemical cell with a positive cell potential, which can allow chemical energy to be converted into electrical energy. More particularly, a galvanic cell can include a first reservoir serving as an anode and a second, dissimilar reservoir serving as a cathode. Each galvanic cell can store chemical potential energy. When a conductive material is located proximate to a cell such that the material can provide electrical and/or ionic communication between the cell elements the chemical potential energy can be released as electrical energy.
  • each set of adjacent, dissimilar reservoirs can function as a single-cell battery, and the distribution of multiple sets of adjacent, dissimilar reservoirs within the apparatus can function as a field of single-cell batteries, which in the aggregate forms a multiple-cell battery distributed across a surface.
  • the galvanic cell can comprise electrodes connected to an external power source, for example a battery or other power source.
  • the electrodes need not comprise dissimilar materials, as the external power source can define the anode and cathode.
  • the power source need not be physically connected to the device.
  • Matrices refer to a pattern or patterns, such as those formed by electrodes on a surface. Matrices can be designed to vary the electric field or electric microcurrent generated. For example, the strength and shape of the field or microcurrent can be altered, or the matrices can be designed to produce an electric field(s) or current of a desired strength or shape.
  • Modulate with regard to enzyme activity or gene expression can refer to any change in the activity of the enzyme or the expression of the gene.
  • modulate can mean to increase, decrease, delay, or accelerate, the activity or expression.
  • Reduction-oxidation reaction or "redox reaction” as used herein refers to a reaction involving the transfer of one or more electrons from a reducing agent to an oxidizing agent.
  • reducing agent can be defined in some embodiments as a reactant in a redox reaction, which donates electrons to a reduced species. A “reducing agent” is thereby oxidized in the reaction.
  • oxidizing agent can be defined in some embodiments as a reactant in a redox reaction, which accepts electrons from the oxidized species. An “oxidizing agent” is thereby reduced in the reaction.
  • a redox reaction produced between a first and second reservoir provides a current between the dissimilar reservoirs.
  • the redox reactions can occur spontaneously when a conductive material is brought in proximity to first and second dissimilar reservoirs such that the conductive material provides a medium for electrical communication and/or ionic communication between the first and second dissimilar reservoirs.
  • electrical currents can be produced between first and second dissimilar reservoirs without the use of an external battery or other power source (e.g., a direct current (DC) such as a battery or an alternating current (AC) power source such as a typical electric outlet).
  • a system is provided which is "electrically self contained,” and yet the system can be activated to produce electrical currents.
  • electrically self contained can be defined in some embodiments as being capable of producing electricity (e.g., producing currents) without an external battery or power source.
  • activated can be defined in some embodiments to refer to the production of electric current through the application of a radio signal of a given frequency or through ultrasound or through electromagnetic induction.
  • a system can be provided which includes an external battery or power source.
  • an AC power source operating at a single or several frequencies can be of any wave form, such as a sine wave, a triangular wave, a trapezoidal wave, or a square wave, or the like.
  • AC power can also be of any frequency such as for example 50 Hz or 60 HZ, or the like.
  • AC power can also be of any voltage, such as for example 1 10 volts, or 220 volts, or the like.
  • an AC power source can be electronically modified, such as for example having the voltage reduced, prior to use.
  • AC voltage can range from micro-volts to several volts.
  • “Stretchable” as used herein refers to the ability of embodiments that stretch without losing their structural integrity. That is, embodiments can stretch to accommodate irregular wound surfaces or surfaces wherein one portion of the surface can move relative to another portion.
  • Wound as used herein includes abrasions, surgical incisions, cuts, punctures, tears, sores, ulcers, blisters, burns, amputations, bites, and any other breach or disruption of superficial tissue such as the skin, mucus membranes, epithelial linings, etc. Disruptions can include inflamed areas, polyps, ulcers, etc. A scar is intended to include hypertrophic scars, keloids, or any healed wound tissue of the afflicted individual. Superficial tissues include those tissues not normally exposed in the absence of a wound or disruption, such as underlying muscle or connective tissue. A wound is not necessarily visible nor does it necessarily involve rupture of superficial tissue, for example a wound can comprise a bacterial infection. Wounds can include insect and animal bites from both venomous and non-venomous insects and animals.
  • Embodiments can produce a complex electric field of varying parameters, such as depths of penetration or strengths.
  • an electric field can be created by passing electrical current through conductors, wherein the electric field is a result of the movement of charge through the conductor. These fields can be manipulated in multiple ways.
  • additional means of generating electric fields can be employed such as use of radio frequency through tissue.
  • Disclosed systems and devices can generate a localized electric field in a pattern determined by the distance between and physical orientation and/or size of the cells or electrodes. Effective depth of the electric field can be predetermined by the orientation and distance between and physical orientation and/or size of the cells or electrodes.
  • the devices can be coated either totally or partially with a hydrogel, or glucose or any other drug, cellular nutrition, stem cells, or other biologic.
  • the electric field can be extended, for example through the use of a hydrogel.
  • certain aspects of the power can be adjusted. For example, the frequency, amplitude, phase, waveform shape, cycle, and pulse duration can be modulated.
  • Embodiments disclosed herein comprise patterns of microcells or reservoirs or dots.
  • the patterns can be designed to produce an electric field, an electric current, or both.
  • the pattern can be designed to produce a specific size, strength, density, shape, or duration of electric field or electric current.
  • reservoir or dot size and separation can be altered.
  • devices disclosed herein can apply an electric field, an electric current, or both wherein the field, current, or both can be of varying size, strength, density, shape, or duration in different areas of tissue.
  • the shapes of the electric field, electric current, or both can be customized, increasing or decreasing very localized watt densities and allowing for the design of "smart patterned electrodes" where the amount of e field over a tissue can be designed or produced or adjusted based on feedback from the tissue or on an algorithm within the sensors and fed-back to a control module.
  • the electric field, electric current, or both can be strong in one zone and weaker in another.
  • the electric field, electric current, or both can change with time and be modulated based on treatment goals or feedback from the tissue or patient.
  • the control module can monitor and adjust the size, strength, density, shape, or duration of electric field or electric current based on tissue parameters.
  • Embodiments disclosed herein comprise biocompatible electrodes or reservoirs or dots on a surface, for example a fabric or the like.
  • the surface can be pliable.
  • the surface can comprise a gauze or mesh.
  • Suitable types of pliable surfaces for use in embodiments disclosed herein can be cloth, absorbent textiles, low-adhesives, vapor permeable films, hydrocolloids, hydrogels, alginates, foams, foam- based materials, cellulose-based materials including Kettenbach fibers, hollow tubes, fibrous materials, such as those impregnated with anhydrous / hygroscopic materials, beads and the like, or any suitable material as known in the art.
  • the pliable material can form, for example, a bandage, a wrist band, a neck band, a waist band, a wound dressing, cloth, fabric, or the like.
  • Embodiments can include coatings on the surface, such as, for example, over or between the electrodes.
  • coatings can include, for example, silicone, and electrolytic mixture, hypoallergenic agents, drugs, biologies, stem cells, skin substitutes, blood coagulants or anti-coagulants, or the like.
  • Drugs suitable for use with embodiments as described herein include analgesics, antibiotics, anti-inflammatories, or the like.
  • the electric field or current produced can "drive" the drug through the skin or surface tissue.
  • Devices herein and placed over tissue such as a joint in motion can move relative to the tissue. Reducing the amount of motion between tissue and dressing can be advantageous to healing. In embodiments, traction or friction blisters can be treated, minimized, or prevented. Slotting or placing strategic cuts into the dressing can make less friction on the skin or tissue. In embodiments, use of an elastic dressing similar to the elasticity of the skin is also possible. The use of the dressing as a temporary bridge to reduce stress across the skin or tissue can reduce stress at the sutures or staples and this will reduce scarring and encourage healing.
  • the material can include a port to access the interior of the material, for example to add fluid, gel, or some other material to the dressing.
  • Certain embodiments can comprise a "blister" top that can enclose a material.
  • the blister top can contain a material that is released into the dressing when the blister is pressed, for example a liquid.
  • the system comprises a component such as elastic to maintain or help maintain its position.
  • the system comprises a component such as an adhesive to maintain or help maintain its position.
  • the adhesive component can be covered with a protective layer that is removed to expose the adhesive at the time of use.
  • the adhesive can comprise, for example, sealants, such as hypoallergenic sealants, gecko sealants, mussel sealants, waterproof sealants such as epoxies, and the like.
  • the positioning component can comprise an elastic film with an elasticity, for example, similar to that of skin, or greater than that of skin, or less than that of skin.
  • the LLEC or LLEF system can comprise a laminate where layers of the laminate can be of varying elasticities.
  • an outer layer may be highly elastic and an inner layers in-elastic.
  • the in-elastic layer can be made to stretch by placing stress relieving discontinuous regions or slits through the thickness of the material so there is a mechanical displacement rather than stress that would break the fabric weave before stretching would occur.
  • the slits can extend completely through a layer or the system or can be placed where expansion is required. In embodiments of the system the slits do not extend all the way through the system or a portion of the system such as the dressing material.
  • the surface can comprise the surface of, for example, a catheter, or a microparticle.
  • a catheter or a microparticle.
  • Such embodiments can be used to treat a subject internally both locally or systemically.
  • the microparticles can be used to make a pharmaceutical composition in combination with a suitable carrier.
  • nanotechnology such as nanobots can be used to provide LLEC systems that can be used as components of pharmaceutical formulations, such as injected, inhaled, or orally administered formulations.
  • a LLEC or LLEF system disclosed herein can comprise "anchor" regions or “arms” to affix the system securely.
  • the anchor regions or arms can anchor the LLEC system, for example to areas around a joint where motion is minimal or limited.
  • a LLEC system can be secured to an area of treatment proximal to a joint, and the anchor regions of the system can extend to areas of minimal stress or movement to securely affix the system.
  • the LLEC system can reduce stress on the area of treatment by "countering" the physical stress caused by movement.
  • a LLEC or LLEF system disclosed herein can comprise reinforcing sections.
  • the reinforcing sections can comprise sections that span the length of the system.
  • a LLEC or LLEF system can comprise multiple reinforcing sections such as at least 1 reinforcing section, at least 2 reinforcing sections, at least 3 reinforcing sections, at least 4 reinforcing sections, at least 5 reinforcing sections, at least 6 reinforcing sections, or the like.
  • the LLEC or LLEF system can comprise additional materials.
  • additional materials can comprise activation gels, rhPDGF (recombinant human platelet- derived growth factor) (REGRANEX ® ), Vibronectin:IGF complexes, CELLSPRAY (Clinical Cell Culture Pty. Ltd., Australia), RECELL ® (Clinical Cell Culture Pty. Ltd., Australia), INTEGRA ® dermal regeneration template (Integra Life Sciences, U.S.), BIOMEND ® (Zimmer Dental Inc., U.S.), INFUSE ® (Medtronic Sofamor Danek Inc., U.S.), ALLODERM ® (LifeCell Corp.
  • activation gels rhPDGF (recombinant human platelet- derived growth factor) (REGRANEX ® ), Vibronectin:IGF complexes, CELLSPRAY (Clinical Cell Culture Pty. Ltd., Australia), RECELL ® (Clinical
  • the additional materials can be, for example, TEGADERM ® 911 10 (3M Corporation, U.S.), MEPILEX ® Normal Gel 0.9% Sodium chloride (Molnlycke Health Care AB, Sweden), HISPAGEL ® (BASF Corporation, U.S.), LUBRIGEL ® (Sheffield Laboratories Corporation, U.S.) or other compositions useful for maintaining a moist environment about an area of treatment or for ease of removal of the LLEC or LLEF system.
  • additional materials that can be added to the LLEC or LLEF system can include for example, vesicular-based formulations such as hemoglobin vesicles.
  • liposome-based formulations can be used.
  • Embodiments can comprise antimicrobial materials, for example gels or liquids.
  • Embodiments can include devices in the form of a gel, such as, for example, a one- or two-component gel that is mixed on use.
  • Embodiments can include devices in the form of a spray, for example, a one- or two- component spray or liquid that is mixed on use.
  • the LLEC or LLEF system can comprise kits which can comprise instructions or directions on how to place the system to maximize its performance.
  • Electrodes or microcells can be or include a conductive metal.
  • the electrodes or microcells can comprise any electrically-conductive material, for example, an electrically conductive hydrogel, metals, electrolytes, superconductors, semiconductors, plasmas, and nonmetallic conductors such as graphite and conductive polymers.
  • Electrically conductive metals can include silver, copper, gold, aluminum, molybdenum, zinc, lithium, tungsten, brass, carbon, nickel, iron, palladium, platinum, tin, bronze, carbon steel, lead, titanium, stainless steel, mercury, Fe/Cr alloys, and the like.
  • the electrode can be coated or plated with a different metal such as aluminum, gold, platinum or silver.
  • reservoir or electrode or cell geometry can comprise circles, polygons, lines, zig-zags, ovals, stars, or any suitable variety of shapes. This provides the ability to design/customize surface electric field shapes as well as depth of penetration.
  • Reservoir or dot sizes and concentrations can be of various sizes, as these variations can allow for changes in the properties of the electric field created. Certain embodiments provide an electric field at about 1 Volt and then, under normal tissue loads with resistance of 100k to 300K ohms, produce a current in the range of 2-10 microamperes. The electric field strength can be determined by calculating 1 ⁇ 2 the separation distance and applying it in the z-axis over the midpoint between the cell. This indicates the theoretical location of the highest strength field line. [064] In certain embodiments dissimilar metals can be used to create an electric field with a desired voltage. In certain embodiments, the pattern of reservoirs can control the watt density and shape of the electric field.
  • in embodiments "ink” or “paint” can comprise any conductive solution suitable for forming an electrode on a surface, such as a conductive metal solution.
  • printing or “painted” can comprise any method of applying a conductive material such as a conductive liquid material to a material upon which a matrix is desired.
  • printing devices can be used to produce LLEC or LLEF systems disclosed herein.
  • inkjet or "3D" printers can be used to produce embodiments.
  • the binders or inks used to produce LLEC or LLEF systems disclosed herein can include, for example, poly cellulose inks, poly acrylic inks, poly urethane inks, silicone inks, and the like.
  • the type of ink used can determine the release rate of electrons from the reservoirs.
  • various materials can be added to the ink or binder such as, for example, conductive or resistive materials can be added to alter the shape or strength of the electric field. Other materials, such as silicon, can be added to enhance scar reduction. Such materials can also be added to the spaces between reservoirs.
  • the power source can be any energy source capable of generating a current in the LLEC system and can include, for example, AC power, DC power, radio frequencies (RF) such as pulsed RF, induction, ultrasound, and the like. Certain embodiments can utilize a power source to create the electric current, such as a battery or a microbattery. In embodiments pulses of current can be employed.
  • RF radio frequencies
  • Dissimilar metals used to make a LLEC or LLEF system disclosed herein can be silver and zinc, and the electrolytic solution can include sodium chloride in water.
  • the electrodes are applied onto a non-conductive surface to create a pattern, most preferably an array or multi-array of voltaic cells that do not spontaneously react until they contact an electrolytic solution, for example wound exudate. Sections of this description use the terms "printing" with "ink,” but it is understood that the patterns may instead be “painted” with “paints.” The use of any suitable means for applying a conductive material is contemplated.
  • in embodiments "ink” or “paint” can comprise any solution suitable for forming an electrode on a surface such as a conductive material including a conductive metal solution.
  • printing or “painted” can comprise any method of applying a solution to a material upon which a matrix is desired. It is also assumed that a competent practitioner knows how to properly apply and cure the solutions without any assistance, other than perhaps instructions that should be included with the selected binder that is used to make the mixtures that will be used in the printing process. [070]
  • a preferred material to use in combination with silver to create the voltaic cells or reservoirs of disclosed embodiments is zinc.
  • Zinc has been well-described for its uses in prevention of infection in such topical antibacterial agents as Bacitracin zinc, a zinc salt of Bacitracin.
  • Zinc is a divalent cation with antibacterial properties of its own in addition to possessing the added benefit of being a cofactor to proteins of the metalloproteinase family of enzymes important to the phagocytic debridement and remodeling phases of wound healing.
  • As a cofactor zinc promotes and accelerates the functional activity of these enzymes, resulting in better more efficient wound healing.
  • primary surface is a surface of a LLEC or LLEF system that comes into direct contact with an area to be treated such as skin surface or a wound.
  • primary surface 2 is one which is desired to be antimicrobial, such as a medical instrument, implant, surgical gown, gloves, socks, table, door knob, or other surface that will contact an electrolytic solution including sweat, so that at least part of the pattern of voltaic cells will spontaneously react and kill bacteria or other microbes.
  • the difference of the standard potentials of the electrodes or dots or reservoirs can be in a range from 0.05 V to approximately 5.0 V.
  • the standard potential can be 0.05 V, 0.06 V, 0.07 V, 0.08 V, 0.09 V, 0.1 V, 0.2 V, 0.3 V, 0.4 V, 0.5 V, 0.6 V, 0.7 V, 0.8 V, 0.9 V, 1 .0 V, 1 .1 V, 1 .2 V, 1 .3 V, 1 .4 V, 1 .5 V, 1 .6 V, 1 .7 V, 1 .8 V, 1.9 V, 2.0 V, 2.1 V, 2.2 V, 2.3 V, 2.4 V, 2.5 V, 2.6 V, 2.7 V, 2.8 V, 2.9 V, 3.0 V, 3.1 V, 3.2 V, 3.3 V, 3.4 V, 3.5 V, 3.6 V, 3.7 V, 3.8 V, 3.9 V, 4.0 V, 4.1 V, 4.2 V, 4.3 V,
  • the difference of the standard potentials of the electrodes or dots or reservoirs can be at least 0.05 V, at least 0.06 V, at least 0.07 V, at least 0.08 V, at least 0.09 V, at least 0.1 V, at least 0.2 V, at least 0.3 V, at least 0.4 V, at least 0.5 V, at least 0.6 V, at least 0.7 V, at least 0.8 V, at least 0.9 V, at least 1 .0 V, at least 1 .1 V, at least 1.2 V, at least 1 .3 V, at least 1 .4 V, at least 1 .5 V, at least 1 .6 V, at least 1 .7 V, at least 1 .8 V, at least 1 .9 V, at least 2.0 V, at least 2.1 V, at least 2.2 V, at least 2.3 V, at least 2.4 V, at least 2.5 V, at least 2.6 V, at least 2.7 V, at least 2.8 V, at least 2.9 V, at least 3.0 V, at
  • the difference of the standard potentials of the electrodes or dots or reservoirs can be not more than 0.05 V, not more than 0.06 V, not more than 0.07 V, not more than 0.08 V, not more than 0.09 V, not more than 0.1 V, not more than 0.2 V, not more than 0.3 V, not more than 0.4 V, not more than 0.5 V, not more than 0.6 V, not more than 0.7 V, not more than 0.8 V, not more than 0.9 V, not more than 1 .0 V, not more than 1 .1 V, not more than 1 .2 V, not more than 1 .3 V, not more than 1 .4 V, not more than 1 .5 V, not more than 1 .6 V, not more than 1 .7 V, not more than 1 .8 V, not more than 1 .9 V, not more than 2.0 V, not more than 2.1 V, not more than 2.2 V, not more than 2.3 V, not more than 2.4 V, not more than 2.5 V
  • LLEC systems can produce a low level micro-current of between for example about 1 and about 200 micro-amperes, between about 10 and about 190 microamperes, between about 20 and about 180 micro-amperes, between about 30 and about 170 micro-amperes, between about 40 and about 160 micro-amperes, between about 50 and about 150 micro-amperes, between about 60 and about 140 micro-amperes, between about 70 and about 130 micro-amperes, between about 80 and about 120 micro-amperes, between about 90 and about 100 micro-amperes, or the like.
  • LLEC systems disclosed herein can produce a low level microcurrent of between for example about 1 and about 10 micro-amperes,
  • LLEC systems can produce a low level micro-current of between for example about 1 and about 400 micro-amperes, between about 20 and about 380 microamperes, between about 400 and about 360 micro-amperes, between about 60 and about 340 micro-amperes, between about 80 and about 320 micro-amperes, between about 100 and about 3000 micro-amperes, between about 120 and about 280 micro-amperes, between about 140 and about 260 micro-amperes, between about 160 and about 240 micro-amperes, between about 180 and about 220 micro-amperes, or the like.
  • LLEC systems can produce a low level micro-current about 10 micro-amperes, about 20 micro-amperes, about 30 micro-amperes, about 40 microamperes, about 50 micro-amperes, about 60 micro-amperes, about 70 micro-amperes, about 80 micro-amperes, about 90 micro-amperes, about 100 micro-amperes, about 1 10 micro-amperes, about 120 micro-amperes, about 130 micro-amperes, about 140 microamperes, about 150 micro-amperes, about 160 micro-amperes, about 170 micro-amperes, about 180 micro-amperes, about 190 micro-amperes, about 200 micro-amperes, about 210 micro-amperes, about 220 micro-amperes, about 240 micro-amperes, about 260 microamperes, about 280 micro-amperes, about 300 micro-
  • LLEC systems can produce a low level micro-current of not more than 10 micro-amperes, or not more than 20 micro-amperes, not more than 30 microamperes, not more than 40 micro-amperes, not more than 50 micro-amperes, not more than 60 micro-amperes, not more than 70 micro-amperes, not more than 80 micro-amperes, not more than 90 micro-amperes, not more than 100 micro-amperes, not more than 1 10 microamperes, not more than 120 micro-amperes, not more than 130 micro-amperes, not more than 140 micro-amperes, not more than 150 micro-amperes, not more than 160 microamperes, not more than 170 micro-amperes, not more than 180 micro-amperes, not more than 190 micro-amperes, not more than 200 micro-amperes, not more than 210 microamperes, not
  • LLEC systems can produce a low level micro-current of not less than 10 micro-amperes, not less than 20 micro-amperes, not less than 30 micro-amperes, not less than 40 micro-amperes, not less than 50 micro-amperes, not less than 60 microamperes, not less than 70 micro-amperes, not less than 80 micro-amperes, not less than 90 micro-amperes, not less than 100 micro-amperes, not less than 1 10 micro-amperes, not less than 120 micro-amperes, not less than 130 micro-amperes, not less than 140 microamperes, not less than 150 micro-amperes, not less than 160 micro-amperes, not less than 170 micro-amperes, not less than 180 micro-amperes, not less than 190 micro-amperes, not less than 200 micro-amperes, not less than 210 micro-amperes,
  • the applied electrodes or reservoirs or dots can adhere or bond to the desired primary surface 2 because a biocompatible binder is mixed, in embodiments into separate mixtures, with each of the dissimilar metals that will create the pattern of voltaic cells, in embodiments. Most inks are simply a carrier, and a binder mixed with pigment. Similarly, conductive metal solutions can be a binder mixed with a conductive element. The resulting conductive metal solutions can be used with an application method such as screen printing to apply the electrodes to the primary surface in predetermined patterns. Once the conductive metal solutions dry and/or cure, the patterns of spaced electrodes can substantially maintain their relative position, even on a flexible material such as that used for a LLEC or LLEF system.
  • the conductive metal solutions can be hand applied onto a common adhesive bandage so that there is an array of alternating electrodes that are spaced about a millimeter apart on the primary surface of the bandage.
  • the solution should be allowed to dry before being applied to a surface so that the conductive materials do not mix, which would destroy the array and cause direct reactions that will release the elements, but fail to simulate the current of injury.
  • silver alone will demonstrate antimicrobial effects, embodiments show antimicrobial activity greater than that of silver alone.
  • the binder itself can have a beneficial effect such as reducing the local concentration of matrix metallo-proteases through an iontophoretic process that drives the cellulose into the surrounding tissue, or through the presence of the electric field. This process can be used to electronically drive other components such as drugs into the surrounding tissue.
  • the binder can include any biocompatible liquid material that can be mixed with a conductive element (preferably metallic crystals of silver or zinc) to create a conductive solution which can be applied as a thin coating to a surface.
  • a conductive element preferably metallic crystals of silver or zinc
  • One suitable binder is a solvent reducible polymer, such as the polyacrylic non-toxic silk-screen ink manufactured by COLORCON ® Inc., a division of Berwind Pharmaceutical Services, Inc. (see COLORCON ® NO-TOX ® product line, part number NT28).
  • the binder is mixed with high purity (at least 99.999%) metallic silver crystals to make the silver conductive solution.
  • the crystals can be of lower purity, for example 99%, or 97%, or 96%, or 95%, or 93%, or 90%, or 88%, or lower.
  • Silver crystals which can be made by grinding silver into a powder, are preferably smaller than 100 microns in size or about as fine as flour.
  • the size of the crystals is about 325 mesh, which is typically about 40 microns in size or a little smaller.
  • the binder is separately mixed with high purity (at least 99.99%, in an embodiment) metallic zinc powder which has also preferably been sifted through standard 325 mesh screen, to make the zinc conductive solution.
  • most of the crystals used should be larger than 325 mesh and smaller than 200 mesh.
  • the crystals used should be between 200 mesh and 325 mesh, or between 210 mesh and 310 mesh, between 220 mesh and 300 mesh, between 230 mesh and 290 mesh, between 240 mesh and 280 mesh, between 250 mesh and 270 mesh, between 255 mesh and 265 mesh, or the like.
  • the size of the metal crystals, the availability of the surface to the conductive fluid and the ratio of metal to binder affects the release rate of the metal from the mixture.
  • about 10 to 40 percent of the mixture should be metal for a longer term bandage (for example, one that stays on for about 10 days).
  • the percent of the mixture that should be metal can be 8 percent, or 10 percent, 12 percent, 14 percent, 16 percent, 18 percent, 20 percent, 22 percent, 24 percent, 26 percent, 28 percent, 30 percent, 32 percent, 34 percent, 36 percent, 38 percent, 40 percent, 42 percent, 44 percent, 46 percent, 48 percent, 50 percent, or the like.
  • a polycellulose ink can be used as a binder.
  • the percent of the mixture that should be metal can be 40 percent, or 42 percent, 44 percent, 46 percent, 48 percent, 50 percent, 52 percent, 54 percent, 56 percent, 58 percent, 60 percent, 62 percent, 64 percent, 66 percent, 68 percent, 70 percent, 72 percent, 74 percent, 76 percent, 78 percent, 80 percent, 82 percent, 84 percent, 86 percent, 88 percent, 90 percent, or the like.
  • polyacrylic ink can crack if applied as a very thin coat, which exposes more metal crystals which will spontaneously react.
  • LLEC or LLEF systems comprising an article of clothing it may be desired to decrease the percentage of metal down to 5 percent or less, or to use a binder that causes the crystals to be more deeply embedded, so that the primary surface will be antimicrobial for a very long period of time and will not wear prematurely.
  • Other binders can dissolve or otherwise break down faster or slower than a polyacrylic ink, so adjustments can be made to achieve the desired rate of spontaneous reactions from the voltaic cells.
  • a pattern of alternating silver masses or electrodes or reservoirs and zinc masses or electrodes or reservoirs can create an array of electrical currents across the primary surface.
  • a basic pattern, shown in FIG. 1 has each mass of silver equally spaced from four masses of zinc, and has each mass of zinc equally spaced from four masses of silver, according to an embodiment.
  • the first electrode 6 is separated from the second electrode 10 by a spacing 8.
  • the designs of first electrode 6 and second electrode 10 are simply round dots, and in an embodiment, are repeated. Numerous repetitions 12 of the designs result in a pattern.
  • each silver design preferably has about twice as much mass as each zinc design, in an embodiment. For the pattern in FIG.
  • the silver designs are most preferably about a millimeter from each of the closest four zinc designs, and vice-versa.
  • the resulting pattern of dissimilar metal masses defines an array of voltaic cells when introduced to an electrolytic solution.
  • a dot pattern of masses like the alternating round dots of FIG. 1 can be preferred when applying conductive material onto a flexible material, such as those used in disclosed embodiments, because the dots won't significantly affect the flexibility of the material.
  • the pattern of FIG. 1 is well suited for general use. To maximize the density of electrical current over a primary surface the pattern 14 of FIG. 2 can be used.
  • the first electrode 6 in FIG. 2 is a large hexagonally shaped dot
  • the second electrode 10 is a pair of smaller hexagonally shaped dots that are spaced from each other.
  • the spacing 8 that is between the first electrode 6 and the second electrode 10 maintains a relatively consistent distance between adjacent sides of the designs.
  • Numerous repetitions 12 of the designs result in a pattern 14 that can be described as at least one of the first design being surrounded by six hexagonally shaped dots of the second design.
  • the pattern 14 of FIG. 2 is well suited for abrasions and burns, as well as for insect bites, including those that can transfer bacteria or microbes or other organisms from the insect. There are of course other patterns that could be printed to achieve similar results.
  • FIGS. 3 and 4 show how the pattern of FIG. 2 can be used to make an adhesive bandage.
  • the pattern shown in detail in FIG. 2 is applied to the primary surface 2 of a material.
  • the back 20 of the printed dressing material is fixed to an absorbent dressing layer 22 such as cotton.
  • the absorbent dressing layer is adhesively fixed to an elastic adhesive layer 16 such that there is at least one overlapping piece or anchor 18 of the elastic adhesive layer that can be used to secure the device.
  • FIG. 5 shows an additional feature, which can be added between designs, that will start the flow of current in a poor electrolytic solution.
  • a fine line 24 is printed using one of the conductive metal solutions along a current path of each voltaic cell.
  • the fine line will initially have a direct reaction but will be depleted until the distance between the electrodes increases to where maximum voltage is realized.
  • the initial current produced is intended to help control edema so that the LLEC system will be effective. If the electrolytic solution is highly conductive when the system is initially applied the fine line can be quickly depleted and the dressing will function as though the fine line had never existed.
  • FIGS. 6 and 7 show alternative patterns that use at least one line design.
  • the first electrode 6 of FIG. 6 is a round dot similar to the first design used in FIG. 1 .
  • the second electrode 10 of FIG. 6 is a line. When the designs are repeated, they define a pattern of parallel lines that are separated by numerous spaced dots.
  • FIG. 7 uses only line designs. The pattern of FIG. 7 is well suited for cuts, especially when the lines are perpendicular to a cut.
  • the first electrode 6 can be thicker or wider than the second electrode 10 if the oxidation-reduction reaction requires more metal from the first conductive element (mixed into the first design's conductive metal solution) than the second conductive element (mixed into the second design's conductive metal solution).
  • the lines can be dashed.
  • Another pattern can be silver grid lines that have zinc masses in the center of each of the cells of the grid.
  • the pattern can be letters printed from alternating conductive materials so that a message can be printed onto the primary surface-perhaps a brand name or identifying information such as patient blood type.
  • the silver design can contain about twice as much mass as the zinc design in an embodiment.
  • each voltaic cell that is in wound fluid can create approximately 1 Volt of potential that will penetrate substantially through the dermis and epidermis. Closer spacing of the dots can decrease the resistance, providing less potential, and the current will not penetrate as deeply. If the spacing falls below about one tenth of a millimeter a benefit of the spontaneous reaction is that which is also present with a direct reaction; silver is electrically driven into the wound, but the current of injury may not be substantially simulated.
  • spacing between the closest conductive materials can be 0.1 mm, or 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1 .1 mm, 1 .2 mm, 1 .3 mm, 1 .4 mm, 1 .5 mm, 1 .6 mm, 1 .7 mm, 1.8 mm, 1 .9 mm, 2 mm, 2.1 mm,
  • spacing between the closest conductive materials can be decreased, for example, to less than 0.01 mm, or 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 00.8 mm, 0.09 mm, .1 mm, 1 mm, 1 .1 mm, 1 .2 mm, 1 .3 mm, 1.4 mm, 1 .5 mm, 1 .6 mm, 1 .7 mm, 1 .8 mm, 1 .9 mm, 2 mm, 2.1 mm, 2.2 mm, 2.3 mm,
  • the density of the conductive materials can be, for example, 20 reservoirs per square inch (/in 2 ), 30 reservoirs /in 2 , 40 reservoirs /in 2 , 50 reservoirs /in 2 , 60 reservoirs /in 2 , 70 reservoirs /in 2 , 80 reservoirs /in 2 , r 90 reservoirs /in 2 , 100 reservoirs /in 2 , 150 reservoirs /in 2 , 200 reservoirs /in 2 , 250 reservoirs /in 2 , 300 reservoirs /in 2 , or 350 reservoirs /in 2 , 400 reservoirs /in 2 , 450 reservoirs /in 2 , 500 reservoirs /in 2 , 550 reservoirs /in 2 , 600 reservoirs /in 2 , 650 reservoirs /in 2 , 700 reservoirs /in 2 , 750 reservoirs /in 2 , more, or the like.
  • the spacing between the closest conductive materials can be not more than 0.1 mm, or not more than 0.2 mm, not more than 0.3 mm, not more than 0.4 mm, not more than 0.5 mm, not more than 0.6 mm, not more than 0.7 mm, not more than 0.8 mm, not more than 0.9 mm, not more than 1 mm, not more than 1 .1 mm, not more than
  • spacing between the closest conductive materials can be not less than 0.1 mm, not less than 0.2 mm, not less than 0.3 mm, not less than 0.4 mm, not less than 0.5 mm, not less than 0.6 mm, not less than 0.7 mm, not less than 0.8 mm, not less than 0.9 mm, not less than 1 mm, not less than 1 .1 mm, not less than 1 .2 mm, not less than 1 .3 mm, not less than 1 .4 mm, not less than 1 .5 mm, not less than 1 .6 mm, not less than 1 .7 mm, not less than 1 .8 mm, not less than 1 .9 mm, not less than 2 mm, not less than 2.1 mm, not less than 2.2 mm, not less than 2.3 mm, not less than 2.4 mm, not less than 2.5 mm, not less than 2.6 mm, not less than 2.7 mm, not less than 2.8 mm
  • the density of the conductive materials can be, for example, more than 20 reservoirs /in 2 , more than 30 reservoirs /in 2 , more than 40 reservoirs /in 2 , more than 50 reservoirs /in 2 , more than 60 reservoirs /in 2 , more than 70 reservoirs /in 2 , more than 80 reservoirs /in 2 , more than 90 reservoirs /in 2 , more than 100 reservoirs /in 2 , more than 150 reservoirs /in 2 , more than 200 reservoirs /in 2 , more than 250 reservoirs /in 2 , more than 300 reservoirs /in 2 , more than 350 reservoirs /in 2 , more than 400 reservoirs /in 2 , more than 450 reservoirs /in 2 , more than 500 reservoirs /in 2 , more than 550 reservoirs /in 2 , more than 600 reservoirs /in 2 , more than 650 reservoirs /in 2 , more than 700 reservoirs /in 2 ,
  • Disclosures of the present specification include LLEC or LLEF systems comprising a primary surface of a pliable material wherein the pliable material is adapted to be applied to an area of tissue; a first electrode design formed from a first conductive liquid that includes a mixture of a polymer and a first element, the first conductive liquid being applied into a position of contact with the primary surface, the first element including a metal species, and the first electrode design including at least one dot or reservoir, wherein selective ones of the at least one dot or reservoir have approximately a 1 .5 mm +/- 1 mm mean diameter; a second electrode design formed from a second conductive liquid that includes a mixture of a polymer and a second element, the second element including a different metal species than the first element, the second conductive liquid being printed into a position of contact with the primary surface, and the second electrode design including at least one other dot or reservoir, wherein selective ones of the at least one other dot or reservoir have approximately a 2.5 mm +/- 2
  • electrodes, dots or reservoirs can have a mean diameter of 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 .0 mm, 1 .1 mm, 1 .2 mm, 1 .3 mm, 1 .4 mm, 1 .5 mm, 1 .6 mm, 1 .7 mm, 1 .8 mm, 1 .9 mm, 2.0 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, 2.5 mm,, 2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm, 3.0 mm, 3.1 mm, 3.2 mm, 3.3 mm, 3.4 mm, 3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3.9 mm, 4.0 mm, 4.1 mm, 4.2 mm, 4.3 mm, 4.4 mm, 4.5 mm,
  • electrodes, dots or reservoirs can have a mean diameter of not less than 0.2 mm, not less than 0.3 mm, not less than 0.4 mm, not less than 0.5 mm, not less than 0.6 mm, not less than 0 .7 mm, not less than 0.8 mm, not less than 0.9 mm, not less than 1 .0 mm, not less than 1 .1 mm, not less than 1 .2 mm, not less than 1 .3 mm, not less than 1 .4 mm, not less than 1 .5 mm, not less than 1 .6 mm, not less than 1 .7 mm, not less than 1 .8 mm, not less than 1 .9 mm, not less than 2.0 mm, not less than 2.1 mm, not less than 2.2 mm, not less than 2 .3 mm, not less than 2.4 mm, not less than 2.5 mm, not less than 2.6 mm, not less than 2 .7 mm
  • electrodes, dots or reservoirs can have a mean diameter of not more than 0.2 mm, not more than 0.3 mm, not more than 0.4 mm, not more than 0.5 mm, not more than 0.6 mm, not more than 0.7 mm, not more than 0.8 mm, not more than 0.9 mm, not more than 1 .0 mm, not more than 1 .1 mm, not more than 1 .2 mm, not more than 1 .3 mm, not more than 1 .4 mm, not more than 1 .5 mm, not more than 1 .6 mm, not more than 1 .7 mm, not more than 1 .8 mm, not more than 1 .9 mm, not more than 2.0 mm, not more than 2.1 mm, not more than 2.2 mm, not more than 2.3 mm, not more than 2.4 mm, not more than 2.5 mm, not more than 2.6 mm, not more than 2.7 mm, not more than 2.8
  • the material concentrations or quantities within and/or the relative sizes (e.g., dimensions or surface area) of the first and second reservoirs can be selected deliberately to achieve various characteristics of the systems' behavior.
  • the quantities of material within a first and second reservoir can be selected to provide an apparatus having an operational behavior that depletes at approximately a desired rate and/or that "dies" after an approximate period of time after activation.
  • the one or more first reservoirs and the one or more second reservoirs are configured to sustain one or more currents for an approximate pre-determined period of time, after activation.
  • the difference of the standard potentials can be substantially less or more.
  • the electrons that pass between the first reservoir and the second reservoir can be generated as a result of the difference of the standard potentials.
  • the voltage present at the site of treatment is typically in the range of millivolts but disclosed embodiments can introduce a much higher voltage, for example near 1 volt when using the 1 mm spacing of dissimilar metals already described. Further, in embodiments utilizing AC power the voltage introduced can vary or cycle over time. In embodiments the higher voltage can drive the current deeper into the treatment area so that dermis and epidermis benefit from the simulated current of injury. In this way the current not only can drive silver and zinc into the treatment area, but the current can also provide a stimulatory current so that the entire surface area can heal simultaneously. In embodiments the current can, for example, kill microbes. In embodiments the electric field can, for example, kill microbes. [0106] Embodiments disclosed herein relating to treatment of diseases or conditions or symptoms can also comprise selecting a patient or tissue in need of, or that could benefit by, treatment of that disease, condition, or symptom.
  • Embodiments can comprise a moisture-sensitive component that changes color when the device is activated and producing an electric current.
  • Certain embodiments include LLEC or LLEF systems comprising dressings or bandages designed to be used on irregular, non-planar, or "stretching" surfaces such as joints.
  • Embodiments disclosed herein can be used with numerous joints of the body, including the jaw, the shoulder, the elbow, the wrist, the finger joints, the hip, the knee, the ankle, the toe joints, etc. Additional embodiments disclosed herein can be used in areas where tissue is prone to movement, for example the eyelid, the ear, the lips, the nose, genitalia, etc.
  • Certain embodiments disclosed herein include a method of manufacturing a substantially planar LLEC or LLEF system, the method comprising joining with a substrate multiple first reservoirs wherein selected ones of the multiple first reservoirs include a reducing agent, and wherein first reservoir surfaces of selected ones of the multiple first reservoirs are proximate to a first substrate surface; and joining with the substrate multiple second reservoirs wherein selected ones of the multiple second reservoirs include an oxidizing agent, and wherein second reservoir surfaces of selected ones of the multiple second reservoirs are proximate to the first substrate surface, wherein joining the multiple first reservoirs and joining the multiple second reservoirs comprises joining using tattooing.
  • the substrate can comprise gauzes comprising dots or electrodes.
  • Embodiments disclosed herein include LLEC and LLEF systems that can produce an electrical stimulus and/or can electro-motivate, electro-conduct, electro-induct, electro- transport, and/or electrophorese one or more therapeutic materials in areas of target tissue (e.g., iontophoresis), and/or can cause one or more biologic or other materials in proximity to, on or within target tissue to be affected (e.g., attract, repel, kill, neutralize, or alter cellular growth/viability/mobility, etc.).
  • target tissue e.g., iontophoresis
  • biologic or other materials e.g., attract, repel, kill, neutralize, or alter cellular growth/viability/mobility, etc.
  • Further disclosure relating to materials that can produce an electrical stimulus can be found in U.S. Patent No. 7,662, 176 entitled FOOTWEAR APPARATUS AND METHODS OF MANUFACTURE AND USE issued February 16, 2010, which is incorporated herein by reference in its entirety.
  • the wound healing process includes several phases, including an inflammatory phase and a proliferative phase.
  • the proliferative phase involves cell migration (such as by human keratinocytes) wherein cells migrate into the wound site and cell proliferation wherein the cells reproduce. This phase also involves angiogenesis and the growth of granulation tissue.
  • cell migration many epithelial cells have the ability to detect electric fields and respond with directed migration. Their response typically requires Ca 2+ influx, the presence of specific growth factors such as Integrin and intracellular kinase activity. Most types of cells migrate directionally in a small electric field, a phenomenon called galvanotaxis or electrotaxis.
  • Treating a wound can include covering the wound with a LLEC or LLEF system to prevent formation, reduce proliferation of, or disrupt an existing biofilm, for example by down regulating gene expression.
  • a wound can be an acute or chronic wound, a diabetic wound of the lower extremities, such as of the legs or feet, a post-radiation tissue injury, crush injuries or compartment syndrome and other acute traumatic ischemias, venous stasis or arterial- insufficiency ulcers, compromised grafts and flaps, infected wounds, pressure ulcers, necrotizing soft-tissue infections, burns, cancer-related wounds, osteomyelitis, surgical wounds, traumatic wounds, insect bites, and the like.
  • a wound can be a non-penetrating wound, such as the result of blunt trauma or friction with other surfaces.
  • a wound can be a penetrating wound. These result from trauma that breaks through the full thickness of skin and include stab wounds (trauma from sharp objects, such as knives), skin cuts, surgical wounds (intentional cuts in the skin to perform surgical procedures), shrapnel wounds (wounds resulting from exploding shells), or gunshot wounds (wounds resulting from firearms).
  • a wound can be a thermal wound such as resulting from heat or cold, a chemical wound such as resulting from an acid or base, an electrical wound, or the like.
  • Chronic wounds often do not heal in normal stages, and the wounds can also fail to heal in a timely fashion.
  • LLEC and LLEF systems disclosed herein can promote the healing of chronic wounds by increasing cell migration, cell proliferation, and/or cell signaling. Increased migration can be seen in various cell types, such as for example keratinocytes.
  • chronic wounds can be treated using methods and devices disclosed herein to prevent the formation of or disrupt an existing biofilm.
  • treating the wound can comprise applying a LLEC or LLEF system to the wound such that the system can stretch with movement of the wound and surrounding area.
  • the system can be stretched before application to the wound such that the wound management system "pulls" the wound edges together.
  • methods for treating or dressing a wound comprises the step of topically administering an additional material on the wound surface or upon the matrix of biocompatible microcells.
  • additional materials can comprise, for example, activation gels, rhPDGF (REGRANEX ® ), Vibronectin:IGF complexes, CELLSPRAY ® , RECELL ® , INTEGRA ® dermal regeneration template, BIOMEND ® , INFUSE ® , ALLODERM ® , CYMETRA ® , SEPRAPACK ® , SEPRAMESH ® , SKINTEMP ® , MEDFIL ® , COSMODERM ® , COSMOPLAST ® , OP-1 ® , ISOLAGEN ® , CARTICEL ® , APLIGRAF ® , DERMAGRAFT ® , TRANSCYTE* ORCEL” 9 , EPICEL ® , and the like.
  • the activation gel can be, for example, TEGADERM ® 91 1 10 by 3M, MEPILEX ® Normal Gel 0.9% Sodium chloride, HISPAGEL ® , LUBRIGEL ® , or other compositions useful for maintaining a moist environment about the wound or useful for healing a wound via another mechanism.
  • Embodiments of the disclosed LLEC and LLEF systems can provide antimicrobial activity.
  • embodiments disclosed herein can prevent, limit, or reduce formation of biofilms, for example by interfering with bacterial signaling. Further embodiments can kill bacteria through an established biofilm.
  • Methods and devices disclosed herein can be used to modulate enzyme activity.
  • embodiments can modulate the activity enzymes that are affected by electric fields or electric currents or both.
  • embodiments disclosed herein can modulate the activity of oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases, and the like.
  • the activity of glycerol-3-phosphate dehydrogenase can be modulated.
  • methods and devices disclosed herein can modulate the activity of co-enzymes.
  • Embodiments can be used to modulate, for example, enzyme activity in mammalian, bacterial, insect, or other cells. Modulation of enzyme activity can include, for example, up- regulation, down-regulation, or the like.
  • Methods and devices disclosed herein can be used to modulate gene expression.
  • methods and devices disclosed herein can be used for reducing expression of quorum sensing genes such as lasR and rhIR.
  • Further embodiments can reduce expression of genes of the redox sensing multidrug efflux system, for example mexAB and mexEF.
  • Embodiments can be used to modulate, for example, gene expression in mammalian, bacterial, insect, or other cells. Modulation of gene expression can include, for example, up- regulation, down-regulation, or the like.
  • Example 1 The following non-limiting examples are provided for illustrative purposes only in order to facilitate a more complete understanding of representative embodiments. These examples should not be construed to limit any of the embodiments described in the present specification including those pertaining to the methods of treating wounds.
  • Example 1
  • biofilms Treatment of biofilms presents a major challenge, because bacteria living within them enjoy increased protection against host immune responses and are markedly more tolerant to antibiotics.
  • Bacteria residing within biofilms are encapsulated in an extracellular matrix, consisting of several components including polysaccharides, proteins and DNA which acts as a diffusion barrier between embedded bacteria and the environment thus retarding penetration of antibacterial agents.
  • the biofilm-residing bacteria are in a physiological state of low metabolism and dormancy increasing their resistance towards antibiotic agents.
  • Chronic wounds present an increasing socio-economic problem and an estimated 1 - 2% of western population suffers from chronic ulcers and approximately 2-4% of the national healthcare budget in developed countries is spent on treatment and complications due to chronic wounds.
  • the incidence of non-healing wounds is expected to rise as a natural consequence of longer lifespan and progressive changes in lifestyle like obesity, diabetes, and cardiovascular disease.
  • Non-healing skin ulcers are often infected by biofilms.
  • Multiple bacterial species reside in chronic wounds; with Pseudomonas aeruginosa, especially in larger wounds, being the most common.
  • P. aeruginosa is suspected to delay healing of leg ulcers.
  • surgical success with split graft skin transplantation and overall healing rate of chronic venous ulcers is presumably reduced when there is clinical infection by P. aeruginosa.
  • P. aeruginosa biofilm is often associated with chronic wound infection.
  • PA01 biofilm was developed in vitro using a polycarbonate filter model. Cells were grown overnight in LB medium at 37°C bacteria were cultured on sterile polycarbonate membrane filters placed on LB agar plates and allowed to form a mature biofilm for 48h. The biofilm was then exposed to BED or placebo for the following 24h.
  • EDS Energy Dispersive X-ray Spectroscopy
  • EDS elemental analysis of the Ag/ZN BED was performed in an environmental scanning electron microscope (ESEM, FEI XL-30) at 25kV. A thin layer of carbon was evaporated onto the surface of the dressing to increase the conductivity.
  • Biofilm was grown on circular membranes and was then fixed in a 4% formaldehyde/2% glutaraldehyde solution for 48 hours at 4°C, washed with phosphate- buffered saline solution buffer, dehydrated in a graded ethanol series, critical point dried, and mounted on an aluminum stub. The samples were then sputter coated with platinum (Pt) and imaged with the SEM operating at 5 kV in the secondary electron mode (XL 30S; FEG, FEI Co., Hillsboro, OR).
  • the LIVE/DEAD BacLight Bacterial Viability Kit for microscopy and quantitative assays was used to monitor the viability of bacterial populations. Cells with a compromised membrane that are considered to be dead or dying stain red, whereas cells with an intact membrane stain green.
  • EPR measurements were performed at room temperature using a Bruker ER 300 EPR spectrometer operating at X-band with a TM 1 10 cavity.
  • the microwave frequency was measured with an EIP Model 575 source-locking microwave counter (EIP Microwave, Inc. , San Jose, CA).
  • the instrument settings used in the spin trapping experiments were as follows: modulation amplitude, 0.32 G; time constant, 0.16 s; scan time, 60 s; modulation frequency, 100 kHz; microwave power, 20 mW; microwave frequency, 9.76 GHz.
  • the samples were placed in a quartz EPR flat cell, and spectra were recorded at ambient temperature (25°C). Serial 1 -min EPR acquisitions were performed.
  • the components of the spectra were identified, simulated, and quantitated as reported.
  • the double integrals of DEPMPO experimental spectra were compared with those of a 1 mM TEMPO sample measured under identical settings to estimate the concentration of superoxide adduct.
  • RNA including the miRNA fraction
  • Norgen RNA isolation kit was used, according to the manufacturer's protocol. Gene expression levels were quantified with real-time PCR system and SYBR Green (Applied Biosystems) and normalized to nadB and proC as housekeeping genes. Expression levels were quantified employing the 2 (-AAct) relative quantification method.
  • the glycerol-3-phosphate dehydrogenase assay was performed using an assay kit from Biovision, Inc. following manufacturer's instructions. Briefly, cells ( ⁇ 1 x 10 6 ) were homogenized with 200 ⁇ ice cold GPDH Assay buffer for 10 minutes on ice and the supernatant was used to measure O.D. and GPDH activity calculated from the results.
  • Control and treated samples were compared by paired t test. Student's t test was used for all other comparison of difference between means. P ⁇ 0.05 was considered significant.
  • Ag/Zn BED disrupts biofilm much better while silver does not have any effect on biofilm disruption.
  • Silver has been recognized for its antimicrobial properties for centuries. Most studies on the antibacterial efficacy of silver, with particular emphasis on wound healing, have been performed on planktonic bacteria. Silver ions, bind to and react with proteins and enzymes, thereby causing structural changes in the bacterial cell wall and membranes, leading to cellular disintegration and death of the bacterium. Silver also binds to bacterial DNA and RNA, thereby inhibiting the basal life processes.
  • FIG. 13 shows PA01 staining of the biofilm demonstrating the lack of elevated mushroom like structures in the Ag/Zn BED treated sample.
  • LasR then, responds to this signal and the LasR:30C12-HSL complex activates transcription of many genes including rhIR, which encodes a second quorum sensing receptor, RhIR which binds to autoinducer C4-HSL produced by Rhll. RhlR:C4- HSL also directs a large regulon of genes.
  • rhIR which encodes a second quorum sensing receptor
  • RhIR which binds to autoinducer C4-HSL produced by Rhll.
  • RhlR:C4- HSL also directs a large regulon of genes.
  • P. aeruginosa defective in QS is compromised in their ability to form biofilms. Quorum sensing inhibitors increase the susceptibility of the biofilms to multiple types of antibiotics.
  • Ag/Zn BED represses the redox sensing multidrug efflux system in P. aeruginosa
  • Ag/Zn BED acts as a reducing agent and reduces protein thiols.
  • One electron reduction of dioxygen 02 results in the production of superoxide anion.
  • Molecular oxygen (dioxygen) contains two unpaired electrons. The addition of a second electron fills one of its two degenerate molecular orbitals, generating a charged ionic species with single unpaired electrons that exhibit paramagnetism.
  • Superoxide anion which can act as a biological reductant and can reduce disulfide bonds, is finally converted to hydrogen peroxide is known to have bactericidal properties.
  • EPR electron paramagnetic resonance
  • Superoxide spin trap was carried out using DEPMPO (2-(diethoxyphosphoryl)-2-methyl-3,4- dihydro-2/-/-pyrrole 1 - oxide) and ⁇ 1 ⁇ superoxide anion production was detected upon 40 mins of exposure to Ag/Zn BED (FIG. 15).
  • DEPMPO diethoxyphosphoryl-2-methyl-3,4- dihydro-2/-/-pyrrole 1 - oxide
  • ⁇ 1 ⁇ superoxide anion production was detected upon 40 mins of exposure to Ag/Zn BED (FIG. 15).
  • MexR and MexT are two multidrug efflux regulators in P. aeruginosa which uses an oxidation-sensing mechanism.
  • MexR and MexT Oxidation of both MexR and MexT results in formation of intermolecular disulfide bonds, which activates them, leading to dissociation from promoter DNA and de-repression of MexAB-oprM and MexEF- oprN respectively, while in a reduced state, they do not transcribe the operons. Induction of Mex operons leads not only to increased antibiotic resistance but also to repression of the quorum sensing cascades and several virulence factors.
  • Glycerol- 3-phosphate dehydrogenase is an enzyme involved in respiration, glycolysis, and phospholipid biosynthesis and is expected to be influenced by external electric fields in P. aeruginosa.
  • We observed significantly diminished glycerol-3-phosphate dehydrogenase enzyme activity by treating P. aeruginosa biofilm to the Ag/Zn BED for 12 hours (n 3).
  • Biofilms formed with Acinetobacter baumannii, Corynebacterium amycolatum, Escherichia coli, Enterobacter aerogenes, Enterococcus faecal is CI 4413, Klebsiella pneumonia, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, and Streptococcus equi clinical isolates were evaluated.
  • BED represses the expression of glyceraldehyde 3-phosphate dehydrogenase. BED also down-regulates the activity of glyceraldehyde 3-phosphate dehydrogenase.
  • drosophila S2 cells are cultured on sterile polycarbonate membrane filters placed on LB agar plates for 48h. The cells are then exposed to BED or placebo for the following 24h. BED represses the expression of insect P450 enzymes. BED also down-regulates the activity of insect P450 enzymes.
  • the in vitro scratch assay is an easy, low-cost and well-developed method to measure cell migration in vitro.
  • the basic steps involve creating a "scratch" in a cell monolayer, capturing images at the beginning and at regular intervals during cell migration to close the scratch, and comparing the images to quantify the migration rate of the cells.
  • the in vitro scratch assay is particularly suitable for studies on the effects of cell-matrix and cell-cell interactions on cell migration, mimic cell migration during wound healing in vivo and are compatible with imaging of live cells during migration to monitor intracellular events if desired.
  • this method has also been adopted to measure migration of individual cells in the leading edge of the scratch.
  • IGF-1 R phosphorylation was demonstrated by the cells plated under the PROCELLERA ® device as compared to cells plated under insulin growth factor alone.
  • Integrin accumulation also affects cell migration. An increase in integrin accumulation was achieved with the LLEC system. Integrin is necessary for cell migration, and is found on the leading edge of migrating cell.
  • the tested LLEC system enhanced cellular migration and IGF-1 R / integrin involvement. This involvement demonstrates the effect that the LLEC system had upon cell receptors involved with the wound healing process.
  • Tissue culture reagents were obtained from Life Technologies UK.
  • the VEGFR inhibitor (catalog number 676475), the PI3K inhibitor LY294002 (catalog number 440202), the Akt inhibitor (catalog number 124005) and the Rho kinase inhibitor Y27632 (catalog number 688001 ) were all obtained from Calbiochem.
  • Rhodamine-phalloidin (E3478) was obtained from Molecular Probes (Leiden, The Netherlands) and anti-tubulin conjugated with FITC was obtained from Sigma.
  • the HUVEC cell line from ATCC was used prior to passage 10.
  • Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) was used for culture cells and EF exposure experiments.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • HUVEC cells were seeded in a trough formed by two parallel (1 cm apart) strips of glass coverslip (No. 1 , length of 22 mm) fixed to the base of the dish with silicone grease. Scratch lines were made perpendicular to the long axis of the chamber with a fine sterile needle and used as reference marks for directed cell migration. Cells were incubated for 24- 48 hours (37°C, 5% C0 2 ) before a roof coverslip was applied and sealed with silicone grease. The final dimensions of the chamber, through which current was passed, were 22x 10x0.2 mm.
  • Agar-salt bridges not less than 15 cm long were used to connect silver/silver-chloride electrodes in beakers of Steinberg's solution (58 mM NaCI, 0.67 mM KCI, 0.44 mM Ca(N0 3 ) 2 , 1 .3 mM MgS0 4 , 4.6 mM Trizma base, pH 7.8-8.0), to pools of excess culture medium at either side of the chamber. Field strengths were measured directly at the beginning of, the end of and during each experiment. No fluctuations in field strength were observed.
  • Oi orientation index
  • a series of images was taken with an image analyser immediately before EF exposure and at 4, 8 and 24 hours of EF exposure.
  • a cell with its long axis parallel to the vector of the EF will have an Oi of -1
  • a cell with its long axis exactly perpendicular to the EF vector will have an Oi of +1.
  • a randomly oriented population of cells will have an average Oi ⁇ defined as [ ⁇ n cos2(a)] ⁇ n ⁇ of 0.
  • the significance of this two- dimensional orientation distribution against randomness was calculated using Rayleigh's distribution.
  • a long:short axis ratio was calculated for assessment of elongation.
  • VEGF165 ELISA kit was obtained from R and D (Minneapolis, MN), and the detailed technical instructions were followed. Confocal microscopy was as described. Statistical analyses were performed using unpaired, two- tailed Student's f-test. Data are expressed as mean ⁇ s.e.m.
  • VEGF activation is a pivotal elements in angiogenic responses and enhanced angiogenesis by electric stimulation in vivo is mediated through VEGFR activation.
  • VEGFR activation we quantified levels of VEGF.
  • EF exposure 200 mV mm "1 , the same as that measured at skin wounds
  • Marked elevation of VEGF in the culture medium was observed as early as 5 minutes after onset of the EF; this was reduced at 1 hour and 2 hours, rose again at 4 hours, and reached a high level by 24 hours.
  • the morphology of the cells treated with VEGFR inhibitor was very similar to control cells. Cells still elongated, although their long axis was slightly reduced, but they were oriented randomly. Inhibition of VEGFRs could conceivably have had detrimental effects on the long-term viability of cells and this could have influenced their orientation responses. To test for this, we compared the orientation response after a short period of inhibitor and EF application. The orientation response was completely abolished at 4 hours and 8 hours in an EF after VEGFR inhibition.
  • the Oi values of the cells treated with VEGFR inhibitor were -0.16 ⁇ 0.05 and -0.05 ⁇ 0.05 in EF for 4 hours and 8 hours, respectively, which is significantly different from the non-inhibitor-treated values of 0.36 ⁇ 0.05 and 0.53 ⁇ 0.05 (P ⁇ 0.01 ).
  • VEGFR activation lead to endothelial cell migration, cell survival and proliferation, which require the activation of Akt, a downstream effectors of PI3K. Both the PI3K inhibitor
  • Rho family of GTPases regulates VEGF-stimulated endothelial cell motility and reorganization of the actin cytoskeleton, which are important in endothelial cell retraction and in the formation of intercellular gaps.
  • the Rho kinase inhibitor, Y27632 decreased the orientation response significantly, with Oi values of 0.55 ⁇ 0.05, 0.45 ⁇ 0.05 and 0.24 ⁇ 0.05 at 10 ⁇ , 20 ⁇ and 50 ⁇ , respectively. Significant Oi values nonetheless remained even at 50 ⁇ , indicating that multiple signaling mechanisms must be involved.
  • Integrins are important in endothelial cell movement and alignment to shear stress and mechanical stimulation.
  • HUVEC cells elongated dramatically in an EF.
  • cells cultured with no EF retained a more-cobblestone-like appearance.
  • Striking cell elongation was induced by a voltage drop of about 0.7-4.0 mV across a cell of -15 ⁇ in diameter.
  • a perfectly round cell has a long:short axis ratio of 1 and, as cells elongate, the ratio increases.
  • Control cells no EF
  • the long:short axis ratio of EF exposed cells indicated gradual cell elongation throughout the 24 hour experimental period.
  • the voltage dependency of the elongation response was more obvious at later times, with a greater long:short axis ratio for cells cultured at higher EFs.
  • the threshold for EF-induced endothelial cell elongation was between 50-75 mV mm "1 , again 0.5-0.75 mV across a cell 10 ⁇ in diameter.
  • the elongation response of endothelial cells was more marked than that seen previously at the same EF strengths, in corneal and lens epithelial cells.
  • VEGFR, PI3K-Akt and Rho signaling are involved in the elongation response
  • the signaling elements required for reorientation are also involved in elongation, but there are subtle differences.
  • the VEGFR inhibitor 50 ⁇ had no effect on the long:short axis ratio of control cells but significantly decreased the long:short axis ratio in EF-treated cells (P ⁇ 0.002).
  • Both the PI3K inhibitor LY294002 and the Akt inhibitor also significantly decreased the long:short axis ratio (both P ⁇ 0.0001 versus control). Cells treated with these drugs elongated less, with LY294002 the more effective in suppressing EF-induced elongation.
  • Endothelial cells migrated directionally toward the anode when cultured in EFs.
  • the directional migration was slow but steady during the EF exposure and was more evident for single cells than for sheets of cells.
  • Cells migrated directionally towards the anode while elongating and reorienting perpendicularly.
  • Lamellipodial extension toward the anode was marked.
  • Directional migration was obvious at a physiological EF strength of 100 mV mm "1 .
  • the threshold field strength that could induce directional migration was therefore below 100 mV mm "1 .
  • Cell migration was quantified as previously and significant anodal migration was evident (P ⁇ 0.0001 ).
  • Migration speed however, remained constant before and after EF exposure, at 1 -2 ⁇ hour "1 , which is significantly slower than most other cell types migrating in an EF.

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Abstract

L'invention concerne un appareil comprenant de multiples premiers réservoirs et de multiples deuxièmes réservoirs reliés à un substrat. Des réservoirs sélectionnés parmi les multiples premiers réservoirs comprennent un agent de réduction et des surfaces de réservoirs sélectionnés parmi les multiples premiers réservoirs sont situées à proximité d'une surface d'un premier substrat. Des réservoirs sélectionnés parmi les multiples deuxièmes réservoirs comprennent un agent d'oxydation et des surfaces de réservoirs sélectionnés parmi les multiples deuxièmes réservoirs sont situées à proximité de la surface du premier substrat.
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US20200197696A1 (en) 2020-06-25
US20150374984A1 (en) 2015-12-31
US10307587B2 (en) 2019-06-04
US20170106188A1 (en) 2017-04-20
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US20190308015A1 (en) 2019-10-10
US20170128720A1 (en) 2017-05-11
US11484708B2 (en) 2022-11-01
EP3151797B1 (fr) 2021-02-17
US20230017025A1 (en) 2023-01-19
US20170113038A1 (en) 2017-04-27
WO2015187858A1 (fr) 2015-12-10

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